Flow cytometry is a powerful tool used for analysis of particles and cells in a myriad of applications primarily in bioscience research and medicine. The analytical strength of the technique is in its ability to parade single particles (including bioparticles such as cells, bacteria and viruses) through a focused spot or spots of light, typically from a laser or lasers, in rapid succession, at rates up to thousands of particles per second. The high photon flux at this focal spot produces scatter of light by a particle and or emission of light from the particle or labels attached to the particle that can be collected and analyzed. This gives the user a wealth of information about individual particles that can be quickly parleyed into statistical information about populations of particles or cells.
In flow cytometry, multi-beam, multi-wavelength excitation is commonly used to increase the available number of fluorophores that can act as optical reporters. The increased spectral space allows for a greater degree of assay multi-plexing for individual targets.
Multi-beam flow cytometry can be implemented in several different ways. The simplest is to co-locate beams along the same optical axis. In this situation, multi-plexing is limited by the spectral overlap of fluorophores excited by the wavelength of the co-located beams. In most systems, beams are delivered to the flow cell in a stacked manner with a small distance between beams. This allows for spatially separated interrogation zones for each laser or other type of light. In these systems, the magnitude of the spatial separation is chosen to reduce cross talk between adjacent lasers while minimizing the uncertainty of particle position due to fluctuations of the fluidic system. As spatial separation increases cross talk decreases, but uncertainty of a particle position increases.
Due to system requirements that mandate high particle analysis rates and high illumination intensity, a small laser spot size is desired at a target. In most systems, a converging beam is required to achieve this level of focus on target. A converging beam is typically produced by expanding laser light (e.g., collimating or partially collimating) and then propagating it through a focusing lens. For systems with multiple lasers, the collimated optical beams may be stacked with a displacement of a few hundred microns to produce a final spot separation in that order. The collimated beams are then passed through a single focusing lens, as seen in FIG. 1, and then propagated to the interrogation zone where the particles pass.
The single lens approach has proven effective and has been a mainstay for many decades. Its drawback is that the focusing lens is coupled to all the beams in the system simultaneously. Adjustment of the single focusing lens or implementing other lens manipulations to improve the focus of one beam degrades the focus of adjacent beams. Such a system can produce lower quality data, necessitate more effort in calibrating the focus, and also makes interchangeability of the light sources (e.g. lasers) extremely difficult.
The solution to these drawbacks, as presented herein, is a system in which converging beams can be propagated through the optical train instead of collimated beams without the introduction of aberrations. See FIG. 2. Such a system will allow for adjustment of each laser beam without interfering with the optical path of adjacent lasers beams and allow for improved data and results.